Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3 - Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety

The chemical structures of six lipid A species (A, B, C, D-1, D-2, and E) p urified from Rhizobium etli CE3 were investigated by one- and two-dimension al NMR spectroscopy. The R. etli lipid A subtypes each contain an unusual a cyloxyacyl residue at position 2' as part of a conserved distal glucosamine moiety but differ in their proximal units. All R. etli lipid A species lac k phosphate groups. However, they are derivatized with an alpha-linked gala cturonic acid group at position 4', as shown by nuclear Overhauser effect s pectroscopy. Component B, which had been not been reported in previous stud ies, features a beta, 1'-6 linked disaccharide of glucosamine acylated at p ositions 2, 3, 2', and 3' in a pattern that is typical of lipid A found in other Gram-negative bacteria. D-1 contains an acylated aminogluconate unit in place of the proximal glucosamine residue of B. C and E lack ester-linke d beta-hydroxyacyl chains at position 3, as judged by their H-3 chemical sh ifts, and may be synthesized from B and D-1, respectively, by the R. etli 3 -O-deacylase. D-2 is an isomer of D-1 that forms nonenzymatically by acyl c hain migration. A may be an elimination product derived from D-1 during hyd rolysis at 100 degrees C (pH 4.5), a step needed to release lipid A from li popolysaccharide. Based on these findings, we propose a biosynthetic scheme for R. etli lipid A in which B is generated first by a variation of the E. coli pathway. The aminogluconate unit of D-1 could then be made from B by enzymatic oxidation of the proximal glucosamine. As predicted by our hypoth esis, enzyme(s) can be demonstrated in extracts of R. etli that convert C-1 4-labeled B to D-1.